Use of hemoglobin in the treatment of hemorrhagic shock

ABSTRACT

A method of treating a mammal suffering from hemorrhagic shock or reducing hypotension secondary to hemorrhagic shock in a mammal suffering from hemorrhagic shock by administering intermolecularly- or intramolecularly-crosslinked stroma-free hemoglobin to the mammal.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.08/819,878, filed on Mar. 18, 1997, which is a divisional of U.S. patentapplication Ser. No. 08/471,847, filed on Jun. 7, 1995, now U.S. Pat.No. 5,614,490, which is a continuation of U.S. patent application Ser.No. 08/237,471, filed on May 3, 1994, now U.S. Pat. No. 5,510,464, whichis a continuation of U.S. patent application Ser. No. 07/828,429, filedon Jan. 30, 1997, now U.S. Pat. No. 5,334,706.

BACKGROUND OF THE INVENTION

The present invention relates to perfusion and specifically to thetherapeutic use of hemoglobin in low doses to increase perfusion.

Perfusion is supplying an organ or tissue with oxygen and nutrients viablood or a suitable fluid. Perfusion is essentially the flow of fluid totissues and organs through arteries and capillaries. Flow may beexpressed as the ratio of pressure to resistance. If adequate oxygen andnutrients are not reaching tissues and organs, therapies to improveperfusion may be employed.

Current management of hypotension, and its concurrent reduction inperfusion of tissues and organs, consists of the administration of (i)vasopressors, (ii) positive inotropic agents, and/or (iii) vascularvolume expanders depending on the underlying etiology. Hypotensionsecondary to actual or relative hypovolemia, which also reducesperfusion, initially is managed by administration of crystalloid orcolloid solutions and/or blood products.

Therapeutics which increase blood pressure are employed in an attempt toincrease perfusion. Vasopressor agents such as epinephrine,phenylephrine, metaraminol and methoxamine cause contraction of themuscles of capillaries and arteries. This increases resistance to theflow of blood and elevates blood pressure. However, these drugs are notoptimal for increasing perfusion. They pose a risk of inducing excessiveblood pressure; are known to cause arrhythmias; require intraarterialpressure monitoring; and tissue sloughing and necrosis may result ifextravasation occurs. Moreover, vasopressor agents may actually decreasethe flow of oxygen and nutrients to tissues and organs. If theconstriction of the capillaries and arteries increases resistance inproportion to the increase in blood pressure, the net flow, i.e.,perfusion, will be unchanged. However, large increases in resistanceresult in decreased flow. At best a localized increase in perfusion inlarge vessels occurs while the flow in capillaries is reduced. Indeed,vasopressor agents have been reported to result in decreased perfusionof vital organs. Moreover, because vasopressors increase venous pressureas well as arterial pressure, and, therefore can limit optimal fluidadministration, such agents generally are given only after sufficientvolume replacement with an appropriate fluid or blood. W. C. Shoemaker,A. W. Fleming, "Resuscitation of the Trauma Patient; Restoration ofHemodynamic Functions Using Clinical Algorithms," Ann. Emerg. Med.,15:1437 (1986). Dopamine hydrochloride is an inotropic agent used in thetreatment of shock to increase blood pressure. It suffers from thedrawbacks noted above for vasopressor agents. In addition, it has a verysmall therapeutic window. Because the dose/response is extremelysensitive, dopamine must be carefully titrated, and invasive monitoring(arterial line) is required.

A class of therapeutics known as plasma expanders or volume replacementsmay be used to increase perfusion where significant blood loss hasoccurred. In this therapy perfusion is increased by administering volumereplacement fluids such as albumin, Ringer's lactate, saline, or dextransolutions. A decrease in blood volume causes a decrease in pressure. Byrestoring the volume, some pressure, and thus flow can be restored. Inaddition, these solutions do not carry oxygen or nutrients. So, whileflow may be restored, oxygen delivery to the tissues is reduced becauseoxygen and nutrient content of the blood is diluted. Hemodilution isbeneficial in that it reduces the viscosity of the blood, thus reducingresistance and increasing flow. But without the necessary oxygen andnutrients, this therapy is not an optimal treatment for significantblood loss.

Volume replacement with whole blood is currently the most efficacioustreatment when there has been significant blood loss. However, thiscannot be used in a pre-hospital setting and its use requires a twentyminute wait for matching and typing, assuming a donor blood supply isavailable. Studies have also shown that the increased viscosityassociated with infusion of blood may limit capillary blood flow. K. M.Jan, J. Heldman, and S. Chen, "Coronary Hemodynamics and OxygenUtilization after Hematocrit Variations in Hemorrhage," Am. J. Physiol.,239:H326 (1980). There is also the risk of viral (hepatitis and/or HIV)or bacterial infection from transfused blood.

Hemoglobin solutions have been under investigation as oxygen carryingplasma expanders or blood substitutes for more than fifty years. Whileno hemoglobin solution is currently approved for use clinically, theyare intended to be used to replace blood lost through hemorrhage. Theireffectiveness as oxygen carriers has been demonstrated. However, theirpotential toxicity has been the focus of much research.

Even small amounts of stroma (cell membrane) in hemoglobin solutionsappear to be toxic. R. W. Winslow, "Hemoglobin-based Red CellSubstitutes," The Johns Hopkins University Press, Baltimore (1992). Suchtoxic effects include renal vasoconstriction and decreased renal flow aswell as hypertension and bradycardia. In 1967 Rabiner utilized rigorouspurification techniques to develop stroma-free hemoglobin which hasprevented some of the toxic effects encountered with prior hemoglobinsolutions.

In connection with toxicity studies for hemoglobin solutions researchersnoted an elevation in blood pressure as early as 1934. W. R. Amberson,J. Flexner, F. R. Steggerada et al., "On Use of Ringer-Locke SolutionsContaining Hemoglobin as a Substitute for Normal Blood in Mammals," J.Cell Comp. Physiol., 5:359 (1934). Current toxicity studies ofhemoglobin solutions continue to note a pressor effect. For example, thereplacement of blood with various bovine hemoglobin solutions in rabbitsin a 1988 study was characterized by significant hemodynamicinstability, with fluctuations of blood pressure and heart rate, andsevere tachypinea. Of the various hemoglobin solutions tested in thisstudy, the purest (which comprised cross-linked hemoglobin) showed theleast toxicity, but nevertheless "did produce a hypertensive reactionsuggestive of a systemic vasoconstrictor effect." M. Feola, J. Simoni,P. C. Canizaro, R. Tran, G. Raschbaum, and F. J. Behal, "Toxicity ofPolymerized Hemoglobin Solutions," Surg. Gynecol. Obstet., 166:211(1988). In 1975 Rabiner reported on the work of a Russian researcher whonoted a beneficial effect following administration of 200-400 ml of a 3%hemoglobin solution heavily contaminated with stroma lipid to each of 20trauma patients, in that there was a stabilization of blood pressure. S.F. Rabiner, "Hemoglobin Solution as a Plasma Expander," Fed. Proc.,34:1454 (1975). In 1949 Amberson et al. reported that the administrationof 2300 ml of a 6% hemoglobin solution (225 grams hemoglobin) restoredblood pressure to normal in a patient who had suffered significant bloodloss through hemorrhage. W. R. Amberson, J. J. Jennings, and C. M.Rhode, "Clinical Experience with Hemoglobin-Saline Solutions," J. Appl.Physiol., 1:469 (1949).

Although methods of measurement and reporting have been inconsistent, anincrease in blood pressure and a fall in heart rate are frequentlyreported findings associated with administration of a variety ofhemoglobin solutions in animals and man. Those researchers noting thepressor effect of solutions include G. A. H. Buttle, A. Kekwick, and A.Schweitzer, "Blood Substitutes in Treatment of Acute Hemorrhage,"Lancet, 2:507 (1940). J. H. Miller and R. K. McDonald, "The Effect ofHemoglobin in Renal Function in the Human," J. Clin. Invest., 30:1033(1951). C. Elia, H. J. Sternberg, A. Greenburg, and G. W. Peskin,"Stroma-free Hemoglobin in the Resuscitation of Hemorrhagic Shock,"Surg. Forum, 25:201 (1974). G. S. Moss, R. l@ DeWoskin, A. L. Rosen, H.Levine, and C. K. Palani, "Transport of Oxygen and Carbon Dioxide byHemoglobin-saline Solution in the Red Cell Free Primate," Surg. Gynecol.Obstet., 142:357 (1976). J. P. Savitsky, J. Doczi, J. Black, and J. D.Arnold, "A Clinical Safety Trial of Stroma-free Hemoglobin," Clin.Pharmacol. Ther., 23:73 (1978). P. E. Keipert and T. M. S. Chang,"Pyridoxylated Polyhemoglobin as a Red Cell Substitute for Resuscitationof Lethal Hemorrhagic Shock in Conscious Rats," Biomater. Med. DevicesArtif. Organs, 13:1 (1985). F. H. Jesch, W. Peters, J. Hobbhahn, M.Schoenberg, and K. Messmer, "Oxygen-transporting Fluids and OxygenDelivery with Hemodilution," Crit. Care Med., 10:270 (1982).

Two early animal studies in which hemoglobin solutions were administeredfollowing controlled hemorrhage and occlusion of the left coronaryartery demonstrated improved myocardial blood flow compared toautologous blood or dextran. G. P. Biro and D. Beresford-Kroeger, "TheEffect of Hemodilution with Stroma-free Hemoglobin and Dextran onCollateral Perfusion of Ischemic Myocardium in the Dog," Am. Hrt. J.,99:64 (1980). M. Feola, D. Azar, and L. Wiener, "Improved Oxygenation ofIschemic Myocardium by Hemodilution with Stroma-free HemoglobinSolution," Chest. 75:369 (1979). Both of these studies were exchangetransfusions (1:1 or 2:1) of very large doses of hemoglobin.

Renal complications frequently have been associated with use of highdoses of hemoglobin solutions. Oliguria and decreased renal flow havebeen a common finding, although improved modifications of hemoglobinappear to have somewhat ameliorated this problem. N. I. Birndorf and H.Lopas, "Effect of Red Cell Stroma-free Hemoglobin Solution on RenalFunction in Monkeys," J. Appl. Physiol., 29:573 (1970). M. Relihan, R.E. Olsen, and M. S. Litwin, "Clearance Rate and Effect on Renal Functionof Stroma-free Hemoglobin Following Renal Ischemia," Ann. Surg., 176:700(1972). Other reactions such as fever, chills, flushing, nausea, andchest and abdominal pain are often experienced.

In sum, hemoglobin solutions at high doses in high volume administeredas oxygen carrying blood substitutes have been reported to increaseblood pressure, and this effect has been characterized alternately astoxic or potentially beneficial.

Applicants have now discovered that, surprisingly, low doses ofhemoglobin in small volumes may be administered therapeutically torapidly increase perfusion.

SUMMARY OF THE INVENTION

This invention provides a method to therapeutically increase perfusionin a mammal comprising administering stroma-free hemoglobin at a doseranging from the least amount effective to increase perfusion, up to adose of about 2500 mg per kilogram of body weight.

DETAILED DESCRIPTION OF THE INVENTION

This invention is the use of low doses of hemoglobin to increaseperfusion in any clinical setting where that positive change isdesirable. This includes the administration of hemoglobin to increaseblood pressure from abnormally low levels, as in shock of hemorrhagic,cardiogenic or septic origin or to increase blood pressure from normallevels to effect improved perfusion, as in stroke therapy.

The hemoglobin should be "stroma-free" as defined by Rabiner et al. inJ. Exp. Med. 126: 1127 to 1142 (1967), and is preferably alpha-alphacrosslinked, prepared by the method described by Przybelski et al. in J.Lab. and Clin. Med. 117: 143-151 (1991). It is preferably human derived,but may be of animal origin or recombinantly produced. It should be in abalanced electrolyte and buffer solution, and preferably is dissolved inone of the plasma expanders such as colloids (plasma, albumin) orcrystalloids (saline, glucose, dextran, gelatins, or Lactated Ringer's).The effect of hemoglobin is independent of the diluent used to make upthe bulk solution. The hemoglobin should be at a concentration of about1 to about 20% in solution, depending upon the application. The doseshould be from about 30 mg per kg of body weight up to about 2500 mg perkg of body weight. The beneficial effect will plateau afteradministration of about 125 mg per kg of body weight. Doses greater thanthis will not enhance the effect, although they will increase theduration of effect.

Conditions in which low dose hemoglobin would be used to rapidlyincrease perfusion would likely be emergent in nature. Such conditionsinclude shock of hemorrhagic, cardiogenic or septic origin. It would beparticularly beneficial in septic shock in which systemic vascularresistance is low causing low blood pressure despite high cardiacoutput. Under these conditions, hemoglobin would be administered as abolus of approximately 100 mg (1.0 ml of 10% hemoglobin per kg) followedby volume expansion with a commonly used crystalloid or colloidsolution.

Use of hemoglobin to maintain adequate perfusion in a critical caresetting would involve slow infusion of a crystalloid/hemoglobin solution30 mg to deliver a minimum of hemoglobin per kg of body weight, whiletitrating to effect. The dose administered should give a rise in meanarterial blood pressure equal to or above normal physiologic levels. Asused herein the term blood pressure shall mean the mean arterial bloodpressure.

This invention has the following advantages over the prior art:

1. It can be administered rapidly (within <1 minute) in small doses (30mg/kg) as a bolus of approximately 75±25 ml or as a continuous infusion,resulting in immediate restoration of blood pressure and perfusion in anadult. This markedly differs from current dose requirements ofcrystalloid solutions of 3 times the volume lost, typicallynecessitating administration of many liters of fluid. Studies in ourlaboratory also indicate that successful resuscitation of hemorrhagicshock can be achieved with hemoglobin solution in one-sixth the dose ofwhole blood.

2. The duration of effect on blood pressure of the lowest dose ofhemoglobin is about 120 minutes, as compared to the transient (30minute) rise in blood pressure achieved by administration of LactatedRinger's, the most commonly used initial resuscitation fluid. Thisshould provide sufficient time to achieve definitive, correctivetreatment.

3. It is preferably stroma-free and, therefore, does not requirecrossmatching or typing. This hastens the time in which definitivetreatment enhancing oxygen delivery can be initiated.

4. It can be purified by heat pasteurization and, therefore, free ofinfective viruses including hepatitis and the human immunodeficiencyvirus. This is not true of blood products.

5. It is hyperoncotic in nature and, thus, increases intravascularvolume. This feature is of particular importance in resuscitation ofpatients in hemorrhagic shock. Recent reports of the improved results ofresuscitation with hyperoncotic saline solutions support the value ofthis additional feature. P. A. Maningas and R. F. Bellamy, "HypertonicSodium Chloride Solutions for the Prehospital Management of TraumaticHemorrhagic Shock: A Possible Improvement in the Standard of Care?",Ann. Emerg. Med., 15:1411 (1986). C. E. Wade, J. P. Hannon, C. A.Bossone, and M. M. Hunt, "Superiority of Hypertonic Saline/Dextran overHypertonic Saline During the First 30 Minutes of Resuscitation FollowingHemorrhagic Hypotension in Conscious Swine," Resuscitation, 20:49(1990). D. S. Prough, J. C. Johnson, D. A. Stump et al., "Effects ofHypertonic Saline Versus Lactated Ringer's Solution on Cerebral OxygenTransport During Resuscitation from Hemorrhagic Shock," J. Neurosurg.,64:627 (1986). J. D. Schmoker, J. Zhuang, and S. R. Shackford,"Hypertonic Fluid Resuscitation Improves Cerebral Oxygen Delivery andReduces Intracranial Pressure After Hemorrhagic Shock," J. Trauma,31:1607 (1991).

6. The magnitude of its effect on blood pressure is non-dose dependentand self-limiting. Both low-doses and high-doses produce a 15-35%increase in mean arterial blood pressure. This important and surprisingcharacteristic of the invention precludes the possibility of an overdoseand the development of dangerous hypertension.

7. It has some oxygen carrying ability thereby increasing oxygendelivery to the tissues, albeit less than the oxygen carrying ability ofhigh doses of hemoglobin solutions used as blood substitutes. However,even the small amount of oxygen carrying hemoglobin provided by thisinvention has a profound beneficial effect when perfusion isconcomitantly increased. Increased oxygen delivery is essential to organviability and is a unique feature of hemoglobin.

8. It has a low viscosity which enhances flow through themicrocirculation, thus preserving organ viability and function.

Variations of hemoglobin, including modified hemoglobin, andintramolecular or intermolecular cross-linked species, may be used inaccordance with this invention to increase perfusion. The effect ofhemoglobin is independent of the diluent used to make up the solution.

Sample guidelines for the clinical administration of hemoglobinsolutions in accordance with this invention for three indications areset forth below:

I. To increase perfusion in conditions of hypoglycemic shock

1. Inject 50 ml (5000 mg) or up to total estimated volume lost of 10%hemoglobin solution (or equivalent) as a bolus into a peripheral IV.

2. Inject hemoglobin solution within the first or "Igolden" hour ofshock state to minimize duration of decreased perfusion.

3. Monitor cuff blood pressure every 15 minutes after administrationuntil peak pressure obtained.

4. Monitor other cardiopulmonary parameters according to standardpractice.

5. Administer other standard therapeutics, as desired or indicated.

II. To increase perfusion in conditions of cardiogenic or septic shock

1. Inject 50 ml (5000 mg) IV bolus, or infuse up to 3000 ml (300 g) of10% hemoglobin solution (or equivalent) at a rate of 1 ml/kg/min toachieve and maintain the desired effect.

2. Administer hemoglobin as early as possible after the development of ashock state to minimize the duration of decreased perfusion.

3. Monitor blood pressure (directly or indirectly) every 15 minutesafter administration until peak pressure obtained.

4. Monitor other cardiopulmonary parameters according to standardpractice.

5. Administer other standard therapeutics, as desired or indicated.

III. To increase perfusion in stroke conditions

1. Infuse at least 100 ml (10 g) of 10% hemoglobin (or equivalent)intravenously at a rate of 1 ml/kg/min to achieve and maintain thedesired effect.

2. Administer hemoglobin solution as early as possible following thecerebrovascular accident to minimize the duration of decreased cerebralperfusion.

3. Monitor blood pressure (directly or indirectly) every 15 minutesafter administration until peak pressure obtained.

4. Monitor other cardiopulmonary parameters according to standardpractice.

5. Administer other standard therapeutics as desired or indicated.

Numerous studies have been performed in our laboratory to determinesafety and efficacy, characterize the pressor response, define optimaldosage, determine modifying factors, and define agents to counteract thepressor effects of hemoglobin. These have been performed as top-loadstudies as well as hemorrhage and exchange-transfusion studies.

EXAMPLE ONE

Toe-load Studies: Safety and Efficacy/Optimal Dose Study

Method: Conscious, unrestrained, male, Sprague-Dawley rats (275-350 g)with previously inserted indwelling arterial and venous catheters wereassigned to one of the following treatment groups:

I. 10% hemoglobin solution at doses of 0.625, 1.25, 2.5, 5.0, 10, 20,and 40 ml/kg (equivalent to 62.5, 125, 250, 500, 1000, 2000, and 4000mg/kg, respectively) (n=6-8)

II. 8.3% human serum albumin (40 ml/kg)

III. 10% hemoglobin solution at doses of 10 ml/kg (1000 mg/kg) and 20ml/kg (2000 mg/kg) intra-arterial OR

10% hemoglobin solution at doses of 10 ml/kg (1000 mg/kg) and 20 ml/kg(2000 mg/kg) intravenous Blood pressure and heart rate were continuouslymonitored for 6 hours after infusion.

Results: 10% hemoglobin at doses from 1.25 to 5 ml/kg (125 to 500 mg/kg)produced an abrupt 25-30% increase in mean arterial pressure (MAP) thatpersisted for 180 minutes. Hemoglobin at 10, 20, and 40 ml/kg (1000,2000, and 4000 mg/kg), likewise, produced an immediate 25-30% increasein MAP that was sustained for 240 to 300 minutes. Although hemoglobin at0.625 ml/kg (62.5 mg/kg) produced a 12% increase in MAP, it was notstatistically significant compared to baseline. Coincident with theincrease in MAP, heart rate (HR) fell in all animals infused withhemoglobin except at the dose of 0.625 ml/kg (62.5 mg/kg). The durationof HR reduction corresponded to the duration of MAP rise. Human serumalbumin (HSA) and Ringer's Lactate (RL) did not change MAP or HRsignificantly compared to baseline. Infusion of hemoglobin 10 ml/kg and20 ml/kg intra-arterially and intravenously resulted in an abruptincrease in MAP and decline in HR that persisted for 240-300 minutes.Statistically, there was no difference between the magnitude andduration of MAP and HR effect between the venous and arterial routes ofadministration. Cardiac output (CO) determinations were performed inonly 2-3 animals receiving hemoglobin at 20 ml/kg or 8.3% HSA (20ml/kg). Although such few animals preclude statistical analysis, COvalues in animals receiving 8.3% HSA rose from a baseline of 30 ml/minto 37 ml/min at the end of infusion. Calculated systemic vascularresistance (SVR) declined from 3200 units at baseline to 2300 units atthe end of infusion. In contrast, CO values in animals receivinghemoglobin 20 ml/kg (2000 mg/kg) declined from baseline of 37 ml/min to34 ml/min at the end of infusion to 26 ml/min by 30 minutespost-infusion. Calculated SVR almost doubled from a baseline of 2600units to 4800 units at the end of infusion, and 4300 units at 30 minutespost-infusion.

Conclusions: Intravenous top-load infusion of up to 40 ml/kg (4000mg/kg) of 10% hemoglobin solution is well tolerated in conscious rats.Doses of hemoglobin between 1.25 and 40 ml/kg (125 and 4000 mg/kg,respectively) elicit a 30-35% increase in MAP that persists between180-300 minutes depending on the volume infused. The lowest dose ofhemoglobin (0.625 ml/kg or 62.5 mg/kg) produced an (12%) increase inMAP. Concomitant reductions in HR of 30-33% from baseline that persistas long as MAP is elevated suggest a baroreceptor reflex response to theabrupt increase in MAP.

EXAMPLE TWO

Characterization of Pressure Response Study

Methods: Conscious, unrestrained, male, Sprague-Dawley rats withindwelling arterial and venous catheters were assigned randomly to oneof four treatment groups:

I. 37° C. 7% hemoglobin 4 ml/kg (280 mg/kg) infused at 0.34 ml/min

II. 37° C. 7% hemoglobin 4 ml/kg (280 mg/kg) as a bolus

III. 4° C. 7% hemoglobin 4 ml/kg (280 mg/kg) infused at 0.34 ml/min

IV. 37° C. 7% hemoglobin 4 ml/kg (280 mg/kg) as a bolus MAP and HR weremonitored continuously throughout the infusion and for 120 minutespost-infusion.

Results: Administration of 7% hemoglobin produced a rapid and sustained(120 minutes) rise in MAP in all treatment groups. However, the maximumpressor response was greatest with warm (37°) vs cold (4°) bolusinjections of hemoglobin (136±4 mmHg vs 119±6 mmHg). A similar, thoughless pronounced response occurred with warm (37°) vs cold (4°) infusionsof hemoglobin (125±5 mmHg vs 118±5 mmHg, respectively). Varying the rateof administration of cold (4°) solution did not alter the pressorresponse significantly. However, the pressor response was attenuatedwith slow infusion (0.34 ml/min) vs bolus administration of warm (37°)hemoglobin (124±5 mmHg vs 134±5 mmHg, respectively). In all cases, HRresponded in a reciprocal manner to the changes in MAP. This reflexresponse was more pronounced (302±11 bpm vs 351±8 bpm) with warm vs coldhemoglobin solution.

The magnitude and duration of the pressor response of 7% hemoglobin (4ml/kg or 280 mg/kg) is affected by the temperature of the solution witha blunted response observed with administration of cold (4°) versus bodytemperature (37°) solution. The rate of administration (bolus vsinfusion at 0.34 ml/min) did not alter the pressor responsesignificantly, regardless of the solution's temperature.

EXAMPLE THREE

Mechanism of Action Study

Methods: Conscious, male, Sprague-Dawley rats were instrumented withindwelling arterial and venous catheters for continuous monitoring ofMAP and HR. The study was divided into two separate sets of experiments:endothelium and nitric oxide/L-NMMA experiments.

Endothelin Study

Animals (1-4 per group) were assigned randomly to receive one of fourtreatments:

I. Big ET (5 nM/kg), IV bolus

II. Phosphoramidon (5 mg/kg), IV bolus

III. Phosphoramidon (5 mg/kg) pretreatment (30 sec) plus Big ET (5nM/kg)

IV. Phosphoramidon (5 mg/kg) pretreatment (30 sec) plus 7% hemoglobin (4ml/kg or 280 mg/kg) IV bolus

Results: 7% hemoglobin (4 ml/kg or 280 mg/kg) elicited a rapid rise inMAP (105±2 mmHg at baseline vs 133±4 mmHg at 15 minutes post-infusion)which peaked at 15 to 25 minutes and returned to baseline at 120minutes. Injection of Big ET (5 nM/kg) elicited a similar, but moredramatic MAP response (98±4 mmHg at baseline vs 149±8 mmHg) which,likewise, peaked at 15 minutes and returned to baseline by 120 minutes.Phosphoramidon, an inhibitor of pro-endothelin conversion to endothelin,given as a top-load injection (5 mg/kg) had no effect on MAP. However,when administered as a 30-sec pre-treatment injection, phosphoramidon (5mg/kg) attenuated the maximum MAP rise of both Big ET (5 nM/kg) and 7%hemoglobin (4 ml/kg or 280 mg/kg) by approximately 75% and 79%,respectively. HR responded reciprocally with MAP with lowest HR'soccurring at maximum MAP. Phosphoramidon, as pre-treatment, alsoattenuated the magnitude of reduction in HR achieved with both ET and 7%hemoglobin.

Nitric Oxide and L-NMMA Study

Animals (5-7 per group) were assigned randomly to receive one of fivetreatments:

I. L-NMMA (5 & 10 mg/kg) IV bolus

II. L-arginine (200 mg/kg) IV bolus

III. L-NMMA (5 mg/kg) plus L-arginine (50 & 100 mg/kg) IV bolus

IV. 7% hemoglobin (4 ml/kg or 280 mg/kg) plus L-arginine (200 mg/kg) IVbolus

V. 7% hemoglobin (4 ml/kg or 280 mg/kg) plus Nitroglycerin infusion(titrated at 10-150 mcg/min to effect) begun 15 minutes post-hemoglobin

Results: L-NMMA injections of 5 and 10 mg/kg increased MAP from 109±3mmHg to 139±13 mmHg and from 106±2 mmHg to 146±6 mmHg, respectively.This response peaked at 30 minutes after injection, and lasted >6 hours.Administration of L-arginine (50 & 100 mg/kg) 30 minutes after injectionof L-NMMA reduced the L-NMMA's pressor effect significantly (p<0.05).Both the magnitude and duration of this attenuation was greater at thehigher dose of L-arginine. Injection of 200 mg/kg of L-arginine innormotensive rats elicited an immediate drop in MAP that quicklyrebounded to above baseline levels within 10 minutes. Injection of thissame dose (200 mg/kg) of L-arginine 15 minutes after a bolus injectionof 7% hemoglobin solution (4 ml/kg or 280 mg/kg) evoked a similar suddenand transient drop in MAP that was followed by an increase in MAP thatexceeded that which would be expected from hemoglobin alone.Nitroglycerin (NTG) infusion (10-150 mcg/min) begun at 15 minutes post7% hemoglobin injection (4 ml/kg or 280 mg/kg) reduced the pressoreffects of hemoglobin, decreasing MAP from a peak of 141±7 mmHg to 113±5mmHG within minutes. Fifteen minutes after discontinuation of NTG, theMAP remained significantly reduced from control values (115±4 mmHg vs128±2 mmHg), respectively.

Conclusions: Hemoglobin solution and Big ET (pro-endothelin) havesimilar pressor effects with respect to MAP peak effect time andduration. However, absolute MAP increase is greater with Big ET than 7%hemoglobin at the doses tested. Phosphoramidon, a metalloproteinaseinhibitor, blunts the pressor effect of both Big ET and hemoglobinsolution, suggesting that the pressor effect is mediated, at least inpart, by ET. Nitroglycerin, a prodrug of nitric oxide (NO), reverseshemoglobin's pressor effects, suggesting that exogenous NO may overridehemoglobin's binding of endogenous NO. However, L-arginine, at a doseexceeding that which reversed the pressor effect of L-NMMA, did notreverse the pressor effects of hemoglobin. This suggests that hemoglobinmay also interfere with the synthesis of NO. Based on these findings, itis concluded that the pressor effects of hemoglobin are mediated, atleast in part, by the release of endothelin (ET), a potentvasoconstrictor, and the inhibition of NO, an endothelin-derivedrelaxing factor. Thus, hemoglobin's pressor effect is mediated by anautoregulatory system which explains the wide margin of safety of thisinvention compared to other pressor agents.

EXAMPLE FOUR

Use of Antihypertensives Agents to Control Pressor Response

Methods: Conscious, unrestrained, male, Sprague-Dawley rats (250-350 g)with indwelling arterial and venous catheters were assigned to one ofthe following five treatment groups, with 6 to 8 animals in each group.MAP and HR were monitored continuously for 120 minutes followinginfusion.

I. 7% hemoglobin (4 ml/kg or 280 mg/kg) IV bolus

II. 7% hemoglobin (4 ml/kg or 280 mg/kg) IV bolus plus Prazosin (2mg/kg, IV over 1 min)

III. 7% hemoglobin (4 ml/kg or 280 mg/kg) IV bolus plus Propranolol (70mcg/kg, IV over 1 min)

IV. 7% hemoglobin (4 ml/kg or 280 mg/kg) IV bolus plus Verapamil (0.25mg/kg, IV over 1 min, repeated in 10 mins)

V. 7% hemoglobin (4 ml/kg or 280 mg/kg) IV bolus plus Nitroglycerin (IVinfusion titrated between 10-150 mcg/min to effect)

Results: 7% hemoglobin infusion elicited an immediate increase in MAPfrom 105±2 mmHg at baseline to 133±4 mmHg at 15 minutes which wassustained for 120 minutes. HR declined in a reciprocal manner. Injectionof Prazosin (2 mg/kg) 15 minutes after injection of hemoglobin, producedan immediate, significant decrease in MAP from a maximum of 134±5 mmHgto 102±11 mmHg with sustained maintenance of MAP near baseline levelsfor one hour. In response to the effect on MAP, HR was restored tobaseline following Prazosin administration, and was sustained throughoutthe 120 minutes observation period.

Administration of Propranolol (70 mcg/kg) 15 minutes after injection ofhemoglobin did not significantly alter its pressor response. An observedbrief (3 to 4 minutes) reduction of MAP immediately followingPropranolol injection did not achieve statistical significance. AlthoughHR returned near baseline levels, it, likewise did not achievestatistical significance.

Verapamil (0.25 mg/kg) transiently decreased MAP from a peak of 143±7mmHg to 118±4 mmHg within 2 minutes of injection. However, MAP returnedto near baseline within 10 minutes. A second bolus injection ofVerapamil produced a similar transient effect. In response, HRtransiently increased toward baseline; however, this did not reachstatistical significance.

Nitroglycerin (NTG) infusion over a dose range of 10 to 150 mcg/kgproduced an immediate and steady decrease in MAP from a peak of 141±7mmHg to 113±5 mmHg. Fifteen minutes after discontinuation of NTG, MAPstill was significantly reduced compared to baseline (115±4 mmHg vs 138±mmHg, respectively). HR returned to baseline by 15 minutes of NTGinfusion and remained at or above baseline for the remainder of theexperiment.

Conclusions: The pressor effects of hemoglobin can be controlled readilywith clinically relevant doses of at least two commonly usedanti-hypertensive agents, Prazosin and Nitroglycerin. The transienteffects of Verapamil on MAP raise the question of whether a higher doseand/or continuous infusion might be more effective. Propranolol, at thedose tested, does not effectively control hemoglobin's pressor effect.

EXAMPLE FIVE

Hemorrhage/Exchange Transfusion Studies

Resuscitation Study

Methods: Male Sprague-Dawley rats were anesthetized with an initial doseof 1.2 ml/kg of a 3:7 mixture of xylazine (20 mg/ml) and ketamine (100mg/ml) and thereafter given 0.6 ml of the same anesthesia solution tomaintain anesthesia. Indwelling arterial and venous catheters andClark-type heated electrodes were placed for continuous monitoring ofMAP, HR and transcutaneous oxygen tension for 60 minutes post-treatment.A sham group was not bled except for withdrawal of two 1 ml bloodsamples, but was monitored the entire period. All other animals werebled a total of 20 ml/kg (approximately one-third total blood volume) ata rate of 1 ml/min. Each rat was assigned randomly to one of sixtreatment groups: (n=5-15 animals per group)

I. Sham

II. No Resuscitation

III. Autologous Shed Blood (20 ml/kg)

IV. Lactated Ringer's 40 ml/kg

V. 14% Hemoglobin 20 ml/kg (2800 mg/kg)

VI. 14% Hemoglobin 10 ml/kg (1400 mg/kg)

All solutions were infused at a rate of 1.7 ml/min.

Results: Following hemorrhage, the MAP fell to 40% of baseline (toapproximately 40 mmHg) in all animals. Within 2 minutes of initiatingresuscitation infusion, whole-volume hemoglobin (20 ml/kg) raised MAP toabove baseline levels (120 mmHg); half-volume hemoglobin (10 ml/kg)raised MAP to baseline levels (100 mmHg); autologous shed blood raisedMAP to approximately 75 mmHg; and Lactated Ringer's raised MAP to 60mmHg. By four minutes both hemoglobin groups had mean arterial pressuressignificantly higher than either the Lactated Ringer's or blood groups.By 6 minutes, there were no differences among the MAPs of the blood,full-volume, and half-volume hemoglobin groups, and all remainedsignificantly higher than the no-resuscitation and Lactated Ringer'sgroups. At 15 minutes post-resuscitation, the MAP of the LactatedRinger's group dropped to the level of the no-resuscitation group. Atthis same time, the MAP of both the whole-volume and half-volumehemoglobin groups were significantly higher than those of the bloodgroup.

HRs in all groups fell during hemorrhage. Within 2 minutes ofresuscitation, HRs in both hemoglobin groups began to rise. By 4 minutesthere were no significant differences in HR among the resuscitatedgroups. However, by 20 minutes, the HRs of the Lactated Ringer's grouphad fallen to the level of the no-resuscitation group, while that of thehemoglobin and blood groups remained near baseline levels.

All animals that were bled had a drop in transcutaneous oxygen tension(TCpO₂) to approximately one-tenth their baseline level. Within 5minutes of resuscitation infusion, all groups, except theno-resuscitation group, had a rise in TCpO₂ to at or near baselinelevels. This trend continued in the blood and hemoglobin groups. Incontrast, a large, persistent drop in TCpO₂ occurred in the LactatedRinger's group which, by 20 minutes, was not significantly differentfrom the no-resuscitation group.

Measurement of serum lactate levels were not significantly different inall groups prior to hemorrhage. However, post-resuscitation serumlactate levels were significantly increased in the Lactated Ringer's andno-resuscitation groups, whereas the sham, blood, whole-volume, andhalf-volume hemoglobin groups had no significant change.

Hematocrit levels measured before and 1 hour after hemorrhage showed asignificant drop in hematocrit in all groups, except for the bloodgroup.

Conclusions: 14% Hemoglobin solution promptly restores MAP, HR, andTCpO₂ after non-lethal hemorrhage. The restoration of TCpO₂ withhemoglobin solution indicates blood flow peripherally and presumably toother organ systems is enhanced. A clinically significant finding isthat half-volume (10 ml/kg) hemoglobin solution is as efficacious inrestoring MAP, HR, and TCpO₂ as nearly twice that volume of whole blood.The return of MAP to baseline before the hemoglobin solution wascompletely infused suggests that even a lower dose of the hemoglobinmight be effective.

EXAMPLE SIX

Hemorrhage/Dose Optimization Study

Methods: Conscious, unrestrained, male, Sprague-Dawley rats (275-300 g)with indwelling venous and arterial catheters were bled 35 ml/kgmanually at 1 ml/min. Twenty minutes after the bleed, the animals wereassigned to one of the following treatment groups:

I. Non-resuscitated control group

II. Autologous shed blood (35 ml/kg)

III. Lactated Ringer's (105 ml/kg) at 3 ml/min

IV. 7% Hemoglobin (17.5 ml/kg=1225 mg/kg OR 35 ml/kg=2450 mg/kg) at 1ml/min

V. 10% Hemoglobin (17.5 ml/kg=1750 mg/kg OR 35 ml/kg=3500 mg/kg) at 1ml/min

HR and pulse pressure were monitored for up to 5 hours.

Results: MAP initially fell 31±3 mmHg following hemorrhage and returnedto 57% of baseline within 20 minutes. This hypotension was associatedwith tachycardia. In the non-resuscitated group, MAP remained at 50 to55 mmHg (from an average baseline of 99.9±4 mmHg) throughout theobservation period, and plummeted just prior to death. At 24 hours, 11out of 15 non-resuscitated animals were dead.

In animals resuscitated with Lactated Ringer's (105 ml/kg), MAPincreased to 80% of baseline during the infusion, but fell to 60 to 70%of baseline at completion of infusion and remained at this levelthroughout the observation period. All animals resuscitated withLactated Ringer's were alive at 24 hours, although all had significanttachycardia.

Animals resuscitated with hemoglobin of either 7% or 10%, and at eitherdose (17.5 ml/kg or 35 ml/kg) as well as animals resuscitated with shedblood had similar hemodynamic responses to resuscitation with anincrease in MAP to near or above baseline levels with a concomitantdecrease in HR. A slightly greater increase in MAP (120 mmHg vs 110mmHg) and a slightly lower HR (350 bpm vs 400 bpm) were noted in theanimals resuscitated with 10% hemoglobin (both doses) at 60 minutespost-infusion. However, at 120 to 300 minutes post-resuscitation, therewere no significant differences between the hemoglobin and blood treatedgroups. At 24 hours post-resuscitation, 4 of 5 blood treated animalswere alive; 8 of 9 animals resuscitated with 10% hemoglobin (17.5 ml/kgor 1750 mg/kg) were alive; 7 of 8 of the 10% hemoglobin (35 ml/kg or3500 mg/kg) group were alive; 3 of 4 of the 7% hemoglobin (17.5 ml/kg or1225 mg/kg) group were alive; and 4 of 5 of the 7% hemoglobin (35 ml/kgor 2450 mg/kg) treated animals were alive.

Conclusions: 7% Hemoglobin solution is as efficacious as a 10%hemoglobin solution in restoring MAP and HR following severe hemorrhage.Furthermore, hemoglobin solution at half the volume (17.5 ml/kg or 1225mg/kg) is as efficacious as blood in restoring cardiovascular functionand increasing survival following hemorrhage.

EXAMPLE SEVEN

Hemorrhage Study

Methods: Conscious, York swine (18-23 kg) with indwelling arterial andvenous thermodilution catheters were bled 30 ml/kg over a 20 minuteperiod and assigned to one of two treatment groups:

I. 7% hemoglobin (10 ml/kg; 700 mg/kg) n=6

II. Autologous shed blood (10 ml/kg) n=6

Following a 2 hour stabilization period, animals received

I. Lactated Ringer's (20 ml/kg)

II. Autologous shed blood (20 ml/kg) Blood samples for buffered baseexcess, hematocrit, and arterial blood gases as well as hemodynamicmeasurements were obtained at baseline, end of hemorrhage, end of firstinfusion, and end of second infusion.

Results: Following hemorrhage, MAP fell 65% from baseline in Group Ianimals, and 62% in Group II animals. SVR fell in both groups.Post-hemorrhage HR in Group I decreased 37% from baseline in contrast toa 4% increase in the shed blood group. This contrasting response isexplained by the significant difference in baseline HR between Group I(198±10 bpm) and Group II (153±10 bpm).

Following administration of hemoglobin (10 ml/kg; 700 mg/kg), MAP roseby 18% (from a baseline of 106±5 mmHg to 125±9 mmHg). This wasaccompanied by a 38% decline in HR (from 198±10 mmHg at baseline to124±5 mmHg), a 10% increase in stroke volume (SV) (from 31 ml/beat atbaseline to 34 ml/beat), and an almost doubling of SVR from 18±1.3 to34±7.1 units.

Following administration of autologous shed blood (10 ml/kg), MAP roseto 96±6 mmHg, but remained 9% below the baseline of 104±8 mmHg. HRremained at control levels and SVR increased from 23.3±2.8 at baselineto 31.4±5.2 units, while SV remained below control values (31 ml/beat vs25 ml/beat).

At 2 hours post-infusion, animals in Group I (hemoglobin) shows MAPsthat remained above baseline, HRs above or close to baseline, continuedelevation of SVR, and a decline in SV to 21 ml/beat. At this same time,animals in Group II (autologous blood) experienced further declines inMAP (86±6 mmHg) and HRs (137±7 bpm). SVR in this group declined, butremained above baseline levels at 27.8±4.1 units with essentially nochange in SV.

Following the infusion of Lactated Ringer's (20 ml/kg), MAP remainedelevated (120±3 mmHg), and HR declined to 160±17 bpm, and SV rose towithin 10% of control levels.

Following infusion of 20 ml/kg autologous blood, MAP rose to near, butstill below, baseline; HR and SVR declined, and SV increased abovebaseline levels.

Analysis of blood samples showed a decrease in venous pH in both groupsfollowing hemorrhage. This value rebounded slightly (from 7.28 to 7.33)following hemoglobin, but remained depressed at 7.28 followingautologous blood. Venous pH returned to normal in both groups followingsupplemental infusion of Lactated Ringer's or autologous blood.

Buffer base excess (BE) dropped significantly in both groups followinghemorrhage, and did not change significantly with infusion of hemoglobinor autologous blood. By 2 hours, BE was returning to baseline in bothgroups, and increased in both groups following final treatment.

Conclusions: 7% Hemoglobin (10 ml/kg or 700 mg/kg) raises blood pressuremore rapidly and to a greater extent than autologous blood, andreplenishes buffer base excess equally well.

EXAMPLE EIGHT

Exchange Transfusion Study I

Methods: Conscious, unrestrained rats were bled a total of 60 ml/kg at arate of 1 ml/min. Infusion of one of the following test solutions wasbegun after the initial 25 ml/kg bleed while animals were bled anadditional 35 ml/kg. (n=8 animals in each group).

I. 7% Hemoglobin (10 ml/kg=700 mg/kg) followed by Lactated Ringer's (50ml/kg) to total volume lost (60 ml/kg)

II. 7% Hemoglobin (20 ml/kg=1400 mg/kg) followed by Lactated Ringer's(120 ml/kg)

III. Lactated Ringer's (180 ml/kg)

All infusions were given at a rate of 1 ml/min until completion ofbleeding and then increased to 3 ml/min. MAP and HR were monitoredcontinuously for 2 hours. Venous blood samples were analyzed for bloodgases, electrolytes, and hematocrit at baseline, end-of-resuscitation,and 1 hour post-resuscitation.

Results: Following the initial bleed of 25 ml/kg, MAP fell toapproximately 30 mmHg. By mid-transfusion (end-of-bleed), MAP wassignificantly higher and near baseline level only in the group receivingthe higher dose (20 ml/kg or 1400 mg/kg) of 7% hemoglobin. This responsewas sustained for the entire observation period (120 minutes). MAP inanimals receiving the lower dose (10 ml/kg or 700 mg/kg) of 7%hemoglobin rose to approximately 70 to 80 mmHg at the end of infusion,and was sustained for 120 minutes. In the Lactated Ringer's group, MAPwas restored to only 60 mmHg (from a baseline of 98 mmHg) at the end ofthe infusion, and thereafter continued to decline with all animals deadwithin 60 to 90 minutes post-infusion. Animals in both hemoglobin groupssurvived longer: 90±9 minutes in the lower dose hemoglobin group and277±50 minutes in the higher dose hemoglobin group.

Blood gas data showed lower HCO₃, pCO₂, and pH levels (metabolicacidosis) in the Lactated Ringer's group compared to the hemoglobintreated groups. Serum K⁺ levels were significantly increased frombaseline in the Lactated Ringer's groups, but were only significantlyincreased at 1 hour post-resuscitation in both hemoglobin groups.

Conclusions: 7% Hemoglobin solution at a dose of 20 ml/kg (1400 mg/kg)followed by 3:1 Lactated Ringer's is superior to lower dose 7%hemoglobin (10 ml/kg or 700 mg/kg) followed by 1:1 Lactated Ringer's,and both hemoglobin doses were superior to 3:1 Lactated Ringer's alone.This study demonstrates that 20 ml/kg or 1400 mg/kg of 7% hemoglobin issufficient for resuscitation following hemorrhage for up to 3 to 4 hoursif adequate crystalloid is infused following hemoglobin administration.

This period of adequate tissue oxygenation may provide the necessary andcritical time before definitive treatment is available.

EXAMPLE NINE

Exchange-Transfusion Study II

Methods: Conscious, unrestrained rats were bled a total of 70 ml/kg(approximately total blood volume) at a rate of 1 ml/min. Infusion ofone of the following test solutions was begun after an initial bleed of35 ml/kg, while the animals continued to be bled an additional 35 ml/kg.(n=6-8 animals per group)

I. 7% Hemoglobin (20 ml/kg=1400 mg/kg) followed by Lactated Ringer's (50ml/kg) to total volume lost of 70 ml/kg

II. Lactated Ringer's (210 ml/kg) to total 3 times the volume lost

III. 5% Human Serum Albumin (HSA) 70 ml/kg

All infusions were given at a rate of 1 ml/min until completion of thebleed, and then increased to 3 ml/min.

Venous blood samples were analyzed for blood gases, electrolytes andhematocrit at baseline, end-of-resuscitation, and 1 hourpost-resuscitation.

Results: MAP fell uniformly to approximately 35 to 40 mmHg at the end ofthe initial 35 ml/kg bleed. Animals transfused with Lactated Ringer'shad a transient increase in MAP (to 50 mmHg) that fell precipitouslyeven before the completion of infusion. HR also remained low during thistreatment but increased significantly by 20 minutes post-resuscitation.By 60 minutes, the one remaining animal in this group had tachycardiawith a HR>450 bpm in response to severe hypotension. Animals transfusedwith HSA had MAPs restored to approximately 60 mmHg. These animals hadconsiderable tachycardia (450-500 bpm) throughout the observationperiod. By 60 minutes, one animal in the HSA group was alive. Animalsresuscitated with 7% hemoglobin had a restoration of MAP to baselineduring and up to 30 minutes post-resuscitation. However, by 60 minutes,only 2 of the hemoglobin treated animals were alive, and their MAPs weredecreasing. HR was rapidly restored to or above baseline for at least 30minutes post-resuscitation. By 60 minutes, HR fell as circulatoryfunction collapsed. Survival time was not significantly differentbetween the Lactated Ringer's and HSA treated animals, but wassignificantly better in the hemoglobin-transfused animals.

All resuscitated animals were extremely acidotic by the end ofresuscitation and at 1 hour post-resuscitation with significant drops inHCO₃, pH and pCO₂. Serum K⁺ levels were significantly elevatedreflecting significant cellular damage from ischemia and hypoxia.

Conclusions: In this more severe transfusion-exchange model, 7%hemoglobin was superior to 3:1 Lactated Ringer's, or isovolume 5% HSA.However, in this model, hemoglobin solution was able to restore andmaintain MAP for only 30 minutes. Although blood gases and chemistrieswere considerably better in the hemoglobin-treated animals, by 1 hourpost-resuscitation animals were decompensating and became asmetabolically acidotic as animals in the other two groups. It ispossible that increasing the volume of Lactated Ringer's (3:1 vs 1:1)following hemoglobin infusion may improve results.

EXAMPLE TEN

Tissue Flow Studs

Methods: Conscious York swine with indwelling venous and rightventricular catheters were bled 30 ml/kg over 30 minutes. Followinghemorrhage, venous blood samples were analyzed for base excess; when BEreached -5 to -10, infusion of 7% hemoglobin 5 ml/kg (350 mg/kg) wasinfused at a rate of 1 ml/kg/min. Animals were monitored for 1 hourpost-infusion at which time they were sacrificed for assessment of organflow.

Results: MAP fell from a mean of 100 mmHg to 40 mmHg followinghemorrhage and promptly returned to baseline following infusion of avery small volume of hemoglobin.

Flow to all organs except the adrenals and the liver, declined followinghemorrhage. Following infusion of 7% hemoglobin, tissue flow increasedto all organ systems except the lung and the liver. Importantly, tissueflow had increased to above baseline levels to the heart and the brain.At 1 hour post-resuscitation, flow to all organs was increased except toparts of the splanchnic system.

Conclusions: Seven percent hemoglobin solution effectively restored MAPfollowing acute hemorrhage. This is associated with an increase inperfusion to vital organ systems, and all other organs with theexception of the lung and the liver. This was achieved with doses as lowas 5 ml/kg (350 mg/kg) or one sixth of the blood volume lost.

EXAMPLE ELEVEN

Cerebral Perfusion Study

Methods: Male, spontaneously hypertensive, anesthetized, andmechanically ventilated rats (350-400 g) with indwelling venous andarterial catheters were assigned randomly to one of the followingtreatment groups: (n=9 animals per group).

I. Hematocrit 44%: blood volume increased with 8 ml donor blood

II. Hematocrit 37%: blood volume and hematocrit manipulated with 8 ml(560 mg) 7% hemoglobin

III. Hematocrit 30%: blood volume and hematocrit manipulated by 5 ml(350 mg) exchange transfusion of 7% hemoglobin plus an additional 8 ml(560 mg) 7% hemoglobin

IV. Hematocrit 23%: blood volume and hematocrit manipulated by 10 ml(700 mg) exchange transfusions of 7% hemoglobin plus an additional 8 ml(560 mg) 7% hemoglobin

V. Hematocrit 16%: blood volume and hematocrit manipulated by 15 ml(1050 mg) exchange transfusion of 7% hemoglobin plus an additional 8 ml(560 mg) 7% hemoglobin

VI. Hematocrit 9%: blood volume and hematocrit manipulated by 20 ml(1400 mg) exchange transfusion plus an additional 8 ml (560 mg) 7%hemoglobin

Maintenance fluids of 0.9 NaCl were infused at 4 ml/kg/hr and targethematocrits and blood volumes maintained for 30 minutes. Via acraniectomy, the middle cerebral artery (MCA) was occluded. After 10minutes of occlusion, 100uCi-kg of C-iodoantipyrine was given. Brainswere then removed, sectioned, and analyzed to define areas with cerebralblood flow (CBF) 0-10 ml/100 g/minute and 11-20 ml/100 g/min.

Results: There was no difference between the hematocrit 44% andhematocrit 37% groups in areas of 0-10 and 11-20 ml/100 g/minute CBF. Inthe other 4 groups, the areas of both of these low CBF's were less ashematocrit decreased, with the smallest area of ischemia occurring inthe hematocrit 9% group (the group that received the largest dose ofhemoglobin). Measurements of CBF in the hemisphere contralateral to theoccluded MCA revealed a progressive increase in CBF as hematocritdecreased (from 125.6±18.8 ml with hematocrit 44% to 180.8±14.4 ml withhematocrit 9%).

Conclusions: Hypervolemic hemodilution with 7% hemoglobin effects adose-related decrease in ischemia following 10 minutes of MCA occlusionin rats. This occurs in association with increased perfusion (CBF)related to increased doses of hemoglobin.

While the foregoing embodiments are intended to illustrate a noveltherapeutic method to increase perfusion, they are not intended norshould they be construed as limitations on the invention. As one skilledin the art would understand, many variations and modifications of theseembodiments may be made which fall within the spirit and scope of thisinvention.

What is claimed is:
 1. A method for treating a mammal suffering fromhemorrhagic shock comprising administering from about 30 to about 1,225milligrams of intermolecularly- or intramolecularly-crosslinkedstroma-free hemoglobin per kilogram of body weight to the mammal.
 2. Themethod of claim 1 wherein from about 30 to about 700 milligrams ofhemoglobin per kilogram of body weight are administered to the mammal.3. The method of claim 1 wherein from about 30 to about 500 milligramsof hemoglobin per kilogram of body weight are administered to themammal.
 4. The method of claim 1 wherein the mammal is a human.
 5. Themethod of claim 1 wherein the hemoglobin is of animal origin.
 6. Themethod of claim 1 wherein the hemoglobin is human derived.
 7. The methodof claim 1 wherein the hemoglobin is recombinantly produced.
 8. Themethod of claim 1 wherein the hemoglobin is administered to the mammalduring emergent care.
 9. A method for reducing hypotension secondary tohemorrhagic shock in a mammal suffering from hemorrhagic shockcomprising administering from about 30 to about 1,225 milligrams ofintermolecularly- or intramolecularly-crosslinked stroma-free hemoglobinper kilogram of body weight to the mammal.
 10. The method of claim 9wherein from about 30 to about 700 milligrams of hemoglobin per kilogramof body weight are administered to the mammal.
 11. The method of claim 9wherein from about 30 to about 500 milligrams of hemoglobin per kilogramof body weight are administered to the mammal.
 12. The method of claim 9wherein the mammal is a human.
 13. The method of claim 9 wherein thehemoglobin is of animal origin.
 14. The method of claim 9 wherein thehemoglobin is human derived.
 15. The method of claim 9 wherein thehemoglobin is recombinantly produced.
 16. The method of claim 9 whereinthe hemoglobin is administered to the mammal during emergent care.
 17. Amethod for treating a mammal suffering from hemorrhagic shock comprisingadministering from about 30 to about 1,225 milligrams of adiaspirin-crosslinked stroma-free hemoglobin per kilogram of body weightto the mammal.
 18. The method of claim 17 wherein from about 30 to about700 milligrams of hemoglobin per kilogram of body weight areadministered to the mammal.
 19. The method of claim 17 wherein fromabout 30 to about 500 milligrams of hemoglobin per kilogram of bodyweight are administered to the mammal.
 20. The method of claim 17wherein the mammal is a human.
 21. The method of claim 17 wherein thehemoglobin is administered to the mammal during emergent care.
 22. Amethod for reducing hypotension secondary to hemorrhagic shock in amammal suffering from hemorrhagic shock comprising administering fromabout 30 to about 1,225 milligrams of a diaspirin-crosslinkedstroma-free hemoglobin per kilogram of body weight to the mammal. 23.The method of claim 22 wherein from about 30 to about 700 milligrams ofhemoglobin per kilogram of body weight are administered to the mammal.24. The method of claim 22 wherein from about 30 to about 500 milligramsof hemoglobin per kilogram of body weight are administered to themammal.
 25. The method of claim 22 wherein the mammal is a human. 26.The method of claim 22 wherein the hemoglobin is administered to themammal during emergent care.